A Novel Role for Vascular Endothelial Growth Factor as an Autocrine Survival Factor for Embryonic Stem Cells during Hypoxia

Vascular endothelial growth factor (VEGF) is best known for its angiogenic activity on endothelial cells, but it also affects neurons, pneumocytes, and other mature cell types as well as endothelial, neural, and hematopoietic progenitors. Here, we examined its effect on pluripotential embryonic stem (ES) cells under hypoxic stress. ES cells were found to produce VEGF and to express VEGF receptor-2 and neuropilin-1 (Nrp-1), a VEGF165 isoform-specific receptor. During hypoxia, expression levels of VEGF, Flk-1, and Nrp-1 were elevated. Inhibition or targeted gene inactivation of VEGF increased ES cell apoptosis during prolonged hypoxia (48 h) by about 10-fold. The survival activity of VEGF was specific since inhibition of other growth factors (including basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, and placental growth factor) had no effect. Neuropilin-1 was involved in the VEGF-survival activity since overexpression of Nrp-1 decreased hypoxia-induced apoptosis about 3-fold. The hypoxia-response element, via which hypoxia-inducible transcription factors up-regulate VEGF expression under hypoxic conditions, was critical since targeted deletion of this element in the VEGF promoter enhanced hypoxia-induced ES cell apoptosis to the same extent as VEGF inhibition or gene inactivation. Thus, VEGF plays a critical role in survival of ES cells during prolonged hypoxia. Vascular endothelial growth factor (VEGF) is best known for its angiogenic activity on endothelial cells, but it also affects neurons, pneumocytes, and other mature cell types as well as endothelial, neural, and hematopoietic progenitors. Here, we examined its effect on pluripotential embryonic stem (ES) cells under hypoxic stress. ES cells were found to produce VEGF and to express VEGF receptor-2 and neuropilin-1 (Nrp-1), a VEGF165 isoform-specific receptor. During hypoxia, expression levels of VEGF, Flk-1, and Nrp-1 were elevated. Inhibition or targeted gene inactivation of VEGF increased ES cell apoptosis during prolonged hypoxia (48 h) by about 10-fold. The survival activity of VEGF was specific since inhibition of other growth factors (including basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, and placental growth factor) had no effect. Neuropilin-1 was involved in the VEGF-survival activity since overexpression of Nrp-1 decreased hypoxia-induced apoptosis about 3-fold. The hypoxia-response element, via which hypoxia-inducible transcription factors up-regulate VEGF expression under hypoxic conditions, was critical since targeted deletion of this element in the VEGF promoter enhanced hypoxia-induced ES cell apoptosis to the same extent as VEGF inhibition or gene inactivation. Thus, VEGF plays a critical role in survival of ES cells during prolonged hypoxia. Blood vessels are critical for delivery of oxygen to growing mammalian tissues. Certain tissues such as the cornea and cartilage in the adult remain avascular and receive oxygen from diffusion of nearby blood vessels, as vessels would impede transmission of light or cause bleeding of joints, respectively. During development, the oocyte in the ovarian follicle receives oxygen through diffusion from perifollicular blood vessels through the follicular fluid, whereas the oxygen needed for cellular metabolism and division of the preimplantation embryo is supplied by diffusion from the fluids within the oviduct and uterus, respectively. Development of the early pre-implantation embryo therefore occurs in an environment with reduced oxygenation until the onset of vascularization after blastocyst implantation (1Blackburn R.O. Clegg E.J. Teratology. 1979; 20: 441-446Crossref PubMed Scopus (6) Google Scholar, 2Graham C.H. Postovit L.M. Park H. Canning M.T. Fitzpatrick T.E. Placenta. 2000; 21: 443-450Crossref PubMed Scopus (86) Google Scholar). The mechanisms responsible for the adaptation of the blastocyst to hypoxic damage remain poorly characterized. A particular growth factor that plays an essential role in the cellular adaptation to hypoxia is vascular endothelial growth factor (VEGF), 1The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; bFGF, basic fibroblast growth factor (FGF); EGF, epidermal growth factor; ES cell, embryonic stem cell; Flk-1, fetal liver kinase-1; Flt-1, fms-like tyrosine kinase-1; HIF, hypoxia-inducible factor; HRE, hypoxia-response element; IGF, insulin-like growth factor; LIF, leukemia inhibitory factor; NRP-1, neuropilin-1; PDGF, platelet-derived growth factor; PlGF, placenta growth factor; WT, wild type. 1The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; bFGF, basic fibroblast growth factor (FGF); EGF, epidermal growth factor; ES cell, embryonic stem cell; Flk-1, fetal liver kinase-1; Flt-1, fms-like tyrosine kinase-1; HIF, hypoxia-inducible factor; HRE, hypoxia-response element; IGF, insulin-like growth factor; LIF, leukemia inhibitory factor; NRP-1, neuropilin-1; PDGF, platelet-derived growth factor; PlGF, placenta growth factor; WT, wild type. one of the most important prototype angiogenic factors in health and disease (3Carmeliet P. Ferreira V. Breier G. Pollefeyt S. Kieckens L. Gertsenstein M. Fahrig M. Vandenhoeck A. Harpal K. Eberhardt C. Declercq C. Pawling J. Moons L. Collen D. Risau W. Nagy A. Nature. 1996; 380: 435-439Crossref PubMed Scopus (3447) Google Scholar, 4Ferrara N. Carver-Moore K. Chen H. Dowd M. Lu L. O'Shea K.S. Powell-Braxton L. Hillan K.J. Moore M.W. Nature. 1996; 380: 439-442Crossref PubMed Scopus (3041) Google Scholar, 5Carmeliet P. Nat. Med. 2003; 9: 653-660Crossref PubMed Scopus (3456) Google Scholar). It is alternatively transcribed in different VEGF isoforms, which bind to VEGF receptor-2 (also named fetal liver kinase-1 (Flk-1) or kinase insert domain-containing receptor (KDR)) and VEGF receptor-1 (also named fms-like tyrosine kinase-1 (Flt-1)) (6Neufeld G. Cohen T. Gitay-Goren H. Poltorak Z. Tessler S. Sharon R. Gengrinovitch S. Levi B.Z. Cancer Metastasis Rev. 1996; 15: 153-158Crossref PubMed Scopus (145) Google Scholar, 7Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3142) Google Scholar). The VEGF165 isoform, which is sufficient for normal vascular development (8Carmeliet P. Ng Y.S. Nuyens D. Theilmeier G. Brusselmans K. Cornelissen I. Ehler E. Kakkar V.V. Stalmans I. Mattot V. Perriard J.C. Dewerchin M. Flameng W. Nagy A. Lupu F. Moons L. Collen D. D'Amore P.A. Shima D.T. Nat. Med. 1999; 5: 495-502Crossref PubMed Scopus (560) Google Scholar, 9Stalmans I. Ng Y.S. Rohan R. Fruttiger M. Bouche A. Yuce A. Fujisawa H. Hermans B. Shani M. Jansen S. Hicklin D. Anderson D.J. Gardiner T. Hammes H.P. Moons L. Dewerchin M. Collen D. Carmeliet P. D'Amore P.A. J. Clin. Investig. 2002; 109: 327-336Crossref PubMed Scopus (422) Google Scholar, 10Stalmans I. Lambrechts D. De Smet F. Jansen S. Wang J. Maity S. von der Kneer P. Ohe M. Swillen A. Maes C. Gewillig M. Molin D.G. Hellings P. Boetel T. Haardt M. Compernolle V. Dewerchin M. Plaisance S. Vlietinck R. Gittenberger-de Emanuel B. Groot A.C. Scambler P. 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VEGF is rapidly and significantly up-regulated in response to hypoxia via activation of the hypoxia-inducible transcription factors HIF-1α and HIF-2α, which bind to the hypoxia-response element (HRE) in the VEGF promotor (14Semenza G.L. Biochem. Pharmacol. 2000; 59: 47-53Crossref PubMed Scopus (444) Google Scholar), leading to angiogenesis and restoration of tissue oxygenation. Recent studies also indicated that VEGF may protect cells against hypoxic damage by directly stimulating their survival, independently of angiogenesis. For instance, VEGF appeared to be critical to prevent motor neuron degeneration, not only because it was essential to maintain sufficient perfusion but also because it exerted direct trophic effects on neuronal survival (15Oosthuyse B. Moons L. Storkebaum E. H. Nuyens D. Brusselmans K. J. Hellings P. M. S. Theilmeier G. Dewerchin M. V. P. H. T. V. L. N. Fujisawa H. R. F. Hicklin D.J. C. P. Lupu F. W. Collen D. Carmeliet P. Nat. PubMed Scopus Google Scholar, D.A. S. A. 2000; PubMed Scopus Google Scholar, M. Storkebaum E. Carmeliet P. Nature. PubMed Scopus Google Scholar). VEGF not only affects cell types but also adult of endothelial and cells, which express Flk-1, and hematopoietic stem cells, which express B. K. S. Trends Med. 2003; 9: Full Text Full Text PDF PubMed Scopus Google Scholar, G. M. M. J. A. J. S. Cancer 2003; Google Scholar, C. C. W. J. PubMed Scopus Google Scholar), we examined in the VEGF also affects embryonic stem (ES) cells, the of the pluripotential cell of ES cells a of for tissue via cellular J. C. A. P. M. 2003; PubMed Scopus Google Scholar, M.T. PubMed Scopus Google Scholar), a of the mechanisms such stem cells against is that ES cells during prolonged hypoxia produce levels of VEGF and its and Nrp-1 via mechanisms the hypoxia-inducible transcription and we a role for VEGF as a survival factor for ES cells in hypoxia. ES and cells in VEGF HIF-1α or were by and in as (3Carmeliet P. Ferreira V. Breier G. Pollefeyt S. Kieckens L. Gertsenstein M. Fahrig M. Vandenhoeck A. Harpal K. Eberhardt C. Declercq C. Pawling J. Moons L. Collen D. Risau W. Nagy A. Nature. 1996; 380: 435-439Crossref PubMed Scopus (3447) Google Scholar, P. Y. D. Brusselmans K. Dewerchin M. M. F. R. P. P. Moons L. Collen D. E. E. Nature. 1998; PubMed Scopus Google Scholar, P. Moons L. A. V. Compernolle V. De M. Y. F. L. H. D. T. T. Dewerchin M. A. Stalmans I. A. S. T. A. W. Hicklin D.J. Collen D. Nat. Med. PubMed Scopus Google Scholar). of the HIF-1α in the VEGF promoter and of ES cells by in in (15Oosthuyse B. Moons L. Storkebaum E. H. Nuyens D. Brusselmans K. J. Hellings P. M. S. Theilmeier G. Dewerchin M. V. P. H. T. V. L. N. Fujisawa H. R. F. Hicklin D.J. C. P. Lupu F. W. Collen D. Carmeliet P. Nat. PubMed Scopus Google Scholar). VEGF, basic fibroblast growth factor platelet-derived growth insulin-like growth epidermal growth factor and PlGF, and against VEGF, bFGF, EGF, or were from inhibitory factor was from and were from and of ES ES cells were in or on embryonic in fetal and ES cells were with and and cell for with in with and fetal ES cells were with and with fetal but was were in an under by the cells in and or under hypoxia by cells in a with and a of for to in or hypoxia for or ES cells were for apoptosis or used for as factors and were to the as of of ES cells was h) during a or with a for of and P. Y. D. Brusselmans K. Dewerchin M. M. F. R. P. P. Moons L. Collen D. E. E. Nature. 1998; PubMed Scopus Google Scholar). ES cells under or hypoxia as were with to the ES cells were and for which was expressed as the of of ES cells was a to the of VEGF, PlGF, and in ES was the indicated by For from cells for was and as (3Carmeliet P. Ferreira V. Breier G. Pollefeyt S. Kieckens L. Gertsenstein M. Fahrig M. Vandenhoeck A. Harpal K. Eberhardt C. Declercq C. Pawling J. Moons L. Collen D. Risau W. Nagy A. Nature. 1996; 380: 435-439Crossref PubMed Scopus (3447) Google Scholar, P. Y. D. Brusselmans K. Dewerchin M. M. F. R. P. P. Moons L. Collen D. E. E. Nature. 1998; PubMed Scopus Google Scholar). were used for VEGF the of the for the for VEGF receptor-1 or of the for VEGF receptor-2 or of the and for neuropilin-1 of the Nrp-1 Neuropilin-1 in ES ES cells were with a the neuropilin-1 gene under of a promoter and the gene as a ES cells with a only the were used as of was by as the of of was from ES cells and with was to the the and and with or and as (8Carmeliet P. Ng Y.S. Nuyens D. Theilmeier G. Brusselmans K. Cornelissen I. Ehler E. Kakkar V.V. Stalmans I. Mattot V. Perriard J.C. Dewerchin M. Flameng W. Nagy A. Lupu F. Moons L. Collen D. D'Amore P.A. Shima D.T. Nat. Med. 1999; 5: 495-502Crossref PubMed Scopus (560) Google for for are expressed as the of were a was as of VEGF and by ES and by wild ES cells were found to produce of VEGF in which were up-regulated the cells were in hypoxia ES cells also the VEGF PlGF, but its expression was not by hypoxia of VEGF was via of HIF-1α to the as ES cells and ES cells with a deletion of the in the VEGF promoter ES were to up-regulate VEGF in response to hypoxia A and by ES cells in in a The survival of VEGF for endothelial cells is known to be via VEGF receptor also or kinase insert domain-containing H.P. A. J. M. V. N. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). that ES cells expressed and that expression of in ES cells was increased in response to hypoxia of in ES cells was since expression levels were in ES cells in and hypoxia hypoxic of (also was in ES cells, whereas expression of PlGF, a was only by hypoxia may the of and a receptor for the VEGF165 is known to as a Y. Oike Y. Ogawa H. Ito Y. Fujisawa H. Suda T. Takakura N. Blood. 2003; 101: 1801-1809Crossref PubMed Scopus (37) Google Scholar). the expression and role of Nrp-1 in ES cell survival was ES cells expressed Nrp-1 in and expression of Nrp-1 was increased in response to hypoxia of Nrp-1 was in ES cells, that HIF-1α was involved ES during VEGF affects survival of ES cells, we the to ES cell apoptosis the of cells was or the of cells was ES cell apoptosis was found to be on the cellular and on the and of hypoxia. in the of LIF, ES cells as by their and growth in cell for LIF, ES cells as of cells with and a ES cells were for in an of ES cells ES cells to apoptosis under oxygen for for the of cells was with and ES cells were for the of cells was with with ES cell apoptosis was also on the of the hypoxic stress. on the that a of ES cells to for we had that of hypoxia would ES cell the ES cells, which were after of hypoxia, to apoptosis for of hypoxia from to was not to ES cell as the for was that a survival the ES cells during prolonged hypoxia. of VEGF in ES during VEGF as a survival factor for cell types and is by hypoxia in ES cells, we examined survival of ES cells under prolonged hypoxia was to the hypoxic of of to the of ES cells, which were under hypoxia for that only survival of ES cells and apoptosis during prolonged hypoxia and h) and against bFGF, EGF, or not apoptosis that the survival activity was by apoptosis of ES cells under hypoxia for was by of by ES cells under hypoxia for or for and that the survival factor VEGF was in the after of hypoxia. only the survival activity in by ES cells under hypoxia for or and that VEGF was the survival factor in the against bFGF, EGF, or not the survival activity in by ES cells under hypoxia for or VEGF165 apoptosis of ES cells during h) and prolonged h) hypoxia, that the survival the in response to hypoxia and the of EGF, or was also ES cells to hypoxia-induced as for VEGF against not survival of ES cells under hypoxic for with by ES cells under hypoxia for whereas also not survival of ES cells under hypoxia with by ES cells under hypoxia for is to to the survival of ES cells during h) or during prolonged hypoxia an to VEGF of ES cells to apoptosis of hypoxia with the that ES cells to apoptosis after of hypoxia that VEGF was only levels VEGF was levels by ES cells and expressed during with its to ES cell apoptosis during hypoxia, expression was not by hypoxia of VEGF in ES during the VEGF apoptosis was in ES cells VEGF ES cells in response to hypoxia but in to ES cells ES cells to apoptosis to of hypoxia, most because not produce the survival factor VEGF The survival activity of VEGF was as ES cells in response to of hypoxia, but ES cells ES cells to hypoxia-induced apoptosis prolonged hypoxia (48 h) The of an effect of on apoptosis in cells was not to expression for a HIF-1α VEGF during ES cells were to up-regulate VEGF in response to hypoxia, with the that hypoxic of VEGF expression is by via of HIF-1α to a specific in the VEGF promoter 2002; 13: PubMed Scopus Google Scholar). 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PubMed Scopus Google Scholar), to the of hypoxic of VEGF in ES cells ES cells during the of hypoxia, and ES cells, to apoptosis during prolonged hypoxia to of Neuropilin-1 in of ES during Nrp-1 plays a role in cell survival, ES cells were with a the gene ES in expression levels in ES cells with a of for ES cells for ES cells, by we that would the survival apoptosis of and ES cells was of hypoxia, a VEGF levels were expression levels the survival response of ES cells in hypoxia, since ES cells were to hypoxic The ES cells after were for cells for ES cells in not and for cells for ES cells in hypoxia of oxygen are for damage and hypoxic cells up-regulate angiogenic factors that the of blood vessels to but mechanisms may the of the cells onset of a that this we that the angiogenic factor VEGF to the survival of ES cells independently of by directly on ES a survival activity of VEGF, via a direct trophic effect on the cell and via an effect on blood vessels, for motor (15Oosthuyse B. 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